Liu Mengying, Hu Yao, Zhu Lijuan, Chen Chen, Zhang Yu, Sun Weixiang, Zhou Qigang
Department of Pharmacology, Pharmacy College, Nanjing Medical University, Nanjing, Jiangsu 210029, China.
J Biomed Res. 2012 Sep;26(5):381-8. doi: 10.7555/JBR.26.20110078. Epub 2012 Apr 18.
We sought to construct the adenoviral vector carrying the gene encoding mouse telomerase reverse transcriptase (mTERT), as well as detect its expression and effect on the proliferation of neuronal stem cells. mTERT was amplified by RT-PCR and then the eukaryotic expression vector of pDC-EGFP-TERT was constructed. After DNA sequence analysis, we detected that there were 293 cells transfected with pDC-EGFP-TERT and helper adenovirus plasmid pBHG lox ΔE1, and three Cre using Lipofectamine 2000 mediation, named Ad-mTERT-GFP, to package adenoviral particles. The Ad-mTERT-GFP was used to infect neuronal stem cells and then the expression and activity of mTERT were detected. In addition, Bromodeoxyuridine labeling test identified the impact of mTERT overexpression on proliferation of neuronal stem cells. The recombinant adenoviral vector confirmed that mTERT was successfully constructed. Overexpression of mTERT stimulated the proliferation of neuronal stem cells both in vitro and in vivo. mTERT overexpression via adenoviral vector carrying mTERT cDNA upregulated the ability of proliferation in neuronal stem cells.
我们试图构建携带编码小鼠端粒酶逆转录酶(mTERT)基因的腺病毒载体,并检测其表达情况以及对神经干细胞增殖的影响。通过逆转录聚合酶链反应(RT-PCR)扩增mTERT,然后构建真核表达载体pDC-EGFP-TERT。经DNA序列分析后,我们使用脂质体2000介导,将pDC-EGFP-TERT和辅助腺病毒质粒pBHG lox ΔE1以及三种Cre转染至293细胞,包装腺病毒颗粒,命名为Ad-mTERT-GFP。用Ad-mTERT-GFP感染神经干细胞,随后检测mTERT的表达及活性。此外,通过溴脱氧尿苷标记试验确定mTERT过表达对神经干细胞增殖的影响。结果证实成功构建了重组腺病毒载体。mTERT的过表达在体外和体内均刺激了神经干细胞的增殖。通过携带mTERT cDNA的腺病毒载体过表达mTERT上调了神经干细胞的增殖能力。